The goal of this project is to determine the extent to which the ribosome exerts control over the fidelity of protein synthesis, by examining ways in which alteration of the structure of the ribosome can influence the level of amino acid substitution errors during protein synthesis in vivo and in vitro. Two approaches are proposed. A) A biochemical approach, which will concentrate on the large ribosomal subunit, is designed to determine whether the fidelity of protein synthesis in vitro can be altered by changes in ribosome structure. The effect of ribosomal proteins on error levels will be examined by means of reconstitution techniques whereby active 50S ribosomal subunits can be constructed which are missing individual non-essential proteins. In addition, chemical modification of ribosomes, and of ribosomal proteins, will be utilized to attempt to create fidelity changes. B) A genetic approach will select mutants of Escherichia coli with increased protein synthesis error levels. The probability of obtaining "ribosomal ambiguity" mutations will be enhanced by the use of mutagenesis localized to the area of the major ribosomal protein gene cluster. The selection will be unbiased with respect to the two ribosomal subunits, unlike previous selections for ribosomal ambiguity, which were strongly biased toward the 30S subunit. The identity of components responsible for such effects will be established by in vitro ribosome reconstitution techniques.